JP6535072B2 - Method for producing biocompatibility polymer porous body, biocompatibility polymer porous body, biocompatibility polymer block and cell structure - Google Patents

Description

  The present invention relates to a method for producing a biocompatible polymer porous body, a biocompatible polymer porous body, a biocompatible polymer block and a cell structure. Specifically, the present invention relates to a biocompatible polymer porous body capable of producing a cell structure exhibiting a high cell number and a high blood vessel number, a method for producing the same, and the biocompatibility polymer porous body The present invention relates to the obtained biocompatible polymer block and cell structure.

  At present, practical application of regenerative medicine for regenerating living tissues and organs that have suffered from dysfunction or malfunction is being promoted. Regenerative medicine is a new medical treatment that re-creates the same form and function as the original tissue by using the three factors of cells, scaffolds and growth factors to restore the living tissue that can not be recovered only by the natural healing ability of the living body. It is a technology. In recent years, treatments using cells are gradually being realized. For example, cultured epidermis using autologous cells, cartilage treatment using autologous chondrocytes, bone regeneration treatment using mesenchymal stem cells, cardiomyocyte sheet treatment using myoblasts, cornea regeneration treatment using a corneal epithelial sheet, The nerve regeneration treatment etc. are mentioned. These new treatments are intended to repair and regenerate living tissue, unlike conventional medicine-based alternative medicine (for example, artificial bone substitutes and hyaluronic acid injection), and provide high therapeutic effects. In fact, products such as cultured epidermis and cultured cartilage using autologous cells have been marketed.

For example, in regeneration of cardiac muscle using a cell sheet, it is considered that a multilayer structure of the cell sheet is necessary to regenerate thick tissue.
Further, Patent Document 1 and Patent Document 2 describe a cell three-dimensional structure produced by three-dimensionally arranging bio-affinitive polymer blocks and cells in a mosaic manner. In this cell three-dimensional structure, nutrient delivery from the outside into the interior of the cell three-dimensional structure is possible, and it has a sufficient thickness and cells are uniformly present in the structure. Moreover, in the example of Patent Document 1, high cell survival activity is demonstrated using a polymer block made of recombinant gelatin and a natural gelatin material, and Patent Document 2 discloses transplantation of a cell three-dimensional structure after transplantation. It is disclosed that blood vessels can be formed at the site.

International Publication WO2011 / 108517 US Patent Application Publication No. 2013/071441

  The three-dimensional structures of cells disclosed in Patent Documents 1 and 2 show high cell survival activity and angiogenic ability, but exhibit higher cell survival activity and angiogenic ability, bioaffinity polymer block and Cell constructs are desired.

  Then, this invention made it the issue which should be solved to provide the biocompatible polymer porous body which makes it possible to provide the cell structure which shows a high cell number and a high blood vessel number, and its manufacturing method. Another object of the present invention is to provide a biocompatible polymer block and a cell structure obtained from the above-mentioned biocompatible polymer porous body.

  As a result of intensive studies to solve the above problems, the inventors of the present invention have found that (a) the difference between the temperature of the highest solution temperature in the solution and the temperature of the lowest solution temperature in the solution is 2.5. And (b) cooling the solution of the biocompatible polymer to an unfrozen state at a temperature not higher than ° C. and the temperature of the portion with the highest liquid temperature in the solution not higher than the melting point of the solvent; A biocompatible polymer porous body produced by the steps of freezing a solution of the obtained biocompatible polymer, and lyophilizing the frozen biocompatible polymer obtained in the step (c) (b) In the above, it was found that the variation in the size of the porous pore is small, that is, the structural uniformity of the porous body is high. Furthermore, the present inventors have found that a cell structure produced using the thus obtained biocompatible high molecular weight porous body exhibits high cell number and high blood vessel number. The present invention has been completed based on these findings.

That is, according to the present invention,
(A) The difference between the temperature of the highest liquid temperature portion in the solution and the temperature of the lowest liquid temperature portion in the solution is 2.5 ° C. or less, and the highest temperature liquid portion in the solution Cooling the solution of the biocompatible polymer at a temperature not higher than the melting point of the solvent to an unfrozen state,
(B) freezing the solution of the biocompatible polymer obtained in the step (a), and (c) lyophilizing the frozen biocompatible polymer obtained in the step (b) A method of producing a biocompatible polymeric porous material is provided.

Preferably, in the step (a), the difference between the temperature of the portion with the highest liquid temperature in the solution and the temperature of the portion with the lowest liquid temperature in the solution immediately before the heat of solidification is 2.5 ° C. or less.
Preferably, in the step (a), the temperature of the lowest temperature part in the solution is a solvent melting point of −5 ° C. or less.

  According to the present invention, there is further provided a biocompatible polymer porous body produced by the method for producing a biocompatible polymer porous body of the present invention.

Further, according to the present invention, the two-dimensional Fourier transform image obtained by applying the two-dimensional Fourier transform to the 1.5 mm square field of view of the cross-sectional structure image of the biocompatible polymer porous body When a line profile is created in which the luminance values around the x-axis coordinate are averaged for the 1/10 pixel width of the image pixel size from the lower end of the y-axis of the Fourier transform image to create a line profile plotted on the y axis A half of the maximum value of is provided with a biocompatible polymer porous body in which no peak is present. The biocompatible polymer porous body is preferably produced by the method for producing a biocompatible polymer porous body of the present invention.
Preferably, assuming that the variance of the base of the line profile is σ, it is assumed that no peak is detected when a peak of 2.0 σ or more is not detected at half of the maximum value of x on the line profile.

  Preferably, the biocompatible polymer is gelatin, collagen, elastin, fibronectin, pronectin, laminin, tenascin, fibrin, fibroin, entactin, thrombospondin, retronectin, polylactic acid, polyglycolic acid, lactic acid / glycolic acid copolymer, hyaluronic acid It is an acid, glycosaminoglycan, proteoglycan, chondroitin, cellulose, agarose, carboxymethylcellulose, chitin or chitosan.

Preferably, the biocompatible polymer is a recombinant gelatin.
Preferably, the recombinant gelatin is
Formula: A-[(Gly-X-Y) n] m-B
(Wherein, A represents any amino acid or amino acid sequence, B represents any amino acid or amino acid sequence, n Xs each independently represent any one of amino acids, n Ys each independently represent an amino acid) N represents an integer of 3 to 100 and m represents an integer of 2 to 10. The n Gly-XY may be the same or different.

Preferably, the recombinant gelatin is
(A) a protein having the amino acid sequence set forth in SEQ ID NO: 1;
(B) a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1 and having biocompatibility; or (c) described in SEQ ID NO: 1 A protein having an amino acid sequence having 80% or more homology with the amino acid sequence of
It is either.

Preferably, the recombinant gelatin is a protein having an amino acid sequence having 90% or more homology with the amino acid sequence set forth in SEQ ID NO: 1 and having biocompatibility.
Preferably, the recombinant gelatin is a protein having an amino acid sequence having 95% or more homology with the amino acid sequence set forth in SEQ ID NO: 1, and having biocompatibility.

  Preferably, the biocompatible polymer in the porous body is crosslinked by heat, ultraviolet light or enzyme.

  According to the present invention, there is further provided a biocompatible polymer block obtained by grinding the biocompatible polymer porous body of the present invention.

  According to the present invention, the biocompatibility polymer block of the present invention and at least one type of cell are further included, and a plurality of biocompatibility polymer blocks are disposed in the gaps between the plurality of cells. Cell structures are provided.

Preferably, the size of one biocompatible polymer block is 20 μm or more and 200 μm or less.
Preferably, the size of one biocompatible polymer block is 50 μm or more and 120 μm or less.

Preferably, the thickness or diameter of the cell structure of the present invention is 400 μm or more and 3 cm or less.
Preferably, the cell structure of the present invention contains 0.0000001 μg or more and 1 μg or less of a biocompatible polymer block per cell.

Preferably, the cell structure of the present invention comprises an angiogenic factor.
Preferably, the cells are cells selected from the group consisting of pluripotent cells, somatic stem cells, progenitor cells and mature cells.
Preferably, the cells are only non-vascular cells.
Preferably, the cells include both non-vascular cells and vasculature cells.

Preferably, in the cell structure, the area of central vasculature cells is larger than the area of peripheral vasculature cells.
Preferably, it has a region in which the percentage of cells in the central vasculature is 60% to 100% of the total area of cells in the vasculature.

Preferably, the cell structure has a region in which the cell density of the central vasculature is 1.0 × 10 ⁻⁴ cells / μm ³ or more.
Preferably, the blood vessel is formed inside the cell structure.

  The cell structure produced by using the biocompatible polymer porous body produced by the method for producing a biocompatible polymer porous body according to the present invention has high cell number and high blood vessel number in the cell structure. Can be shown. According to the cell structure provided by the present invention, more effective cell transplantation therapy can be performed.

FIG. 1 shows the liquid temperature profile of the experiment described in “A”. FIG. 2 shows the liquid temperature profile of the experiment described in “A”. FIG. 2 shows the liquid temperature profile of the experiment described in “U”. FIG. 4 shows the liquid temperature profile of the experiment described in “D”. FIG. 5 shows the liquid temperature profile of the experiment described in "O". FIG. 6 shows the liquid temperature profile of the experiment described in “K”. FIG. 7 shows the liquid temperature profile of the experiment described in "A-a". FIG. 8 shows the liquid temperature profile of the experiment described in “Good”. FIG. 9 shows the uniformity evaluation (two-dimensional Fourier transform and axial power spectrum) of the CBE3 porous body obtained in “A” and “A”. FIG. 10 shows the uniformity evaluation (two-dimensional Fourier transform and axial power spectrum) of the CBE3 porous body obtained in “C” and “D”. FIG. 11 shows the uniformity evaluation (two-dimensional Fourier transform and axial power spectrum) of the CBE3 porous body obtained by “O” and “K”. FIG. 12 shows the uniformity evaluation (two-dimensional Fourier transform and axial power spectrum) of the CBE3 porous body obtained by "A" and "B". FIG. 13 shows the evaluation of cell number and blood vessels in hMSC mosaic cell mass using differentially large blocks A and B. FIG. 14 shows the evaluation of cell number and blood vessels in hMSC mosaic cell mass using the large temperature differential block C. FIG. 15 shows the evaluation of cell number and blood vessels in hMSC mosaic cell mass using small temperature blocks E and F. FIG. 16 shows the evaluation of cell number and blood vessels in hMSC + hECFC mosaic cell mass using differentially large blocks A and B. FIG. 17 shows the evaluation of cell number and blood vessels in hMSC + hECFC mosaic cell mass using small temperature blocks E and F.

Hereinafter, embodiments of the present invention will be described in detail.
(1) Method of Producing Biocompatible Polymeric Porous Body The method of producing the biocompatible polymeric porous body of the present invention is
(A) The difference between the temperature of the highest liquid temperature portion in the solution and the temperature of the lowest liquid temperature portion in the solution is 2.5 ° C. or less, and the highest temperature liquid portion in the solution Cooling the solution of the biocompatible polymer at a temperature not higher than the melting point of the solvent to an unfrozen state,
(B) freezing the solution of the biocompatible polymer obtained in the step (a), and (c) lyophilizing the frozen biocompatible polymer obtained in the step (b).

In the method of the present invention, when the solution of the biocompatible polymer is cooled to the unfrozen state, the difference between the temperature of the highest temperature portion and the temperature of the lowest temperature portion in the solution is 2. By reducing the temperature difference to 5 ° C. or less (preferably 2.3 ° C. or less, more preferably 2.1 ° C. or less), the variation in the size of the obtained porous pores is reduced. The lower limit of the difference between the temperature of the highest liquid temperature portion and the temperature of the lowest liquid temperature portion in the solution is not particularly limited and may be 0 ° C. or more, for example, 0.1 ° C. or more, 0.5 It may be 0 ° C. or more, 0.8 ° C. or more, or 0.9 ° C. or more. Thereby, the cell structure using the biocompatible polymer block produced by using the porous body produced by the method of the present invention achieves the effect of showing high cell number and high blood vessel number.
The cooling in the step (a) is preferably performed, for example, through a material having a thermal conductivity lower than that of water (preferably, Teflon (registered trademark)), and the highest temperature portion in the solution is cooled. The portion farthest from the side can be simulated, and the portion with the lowest liquid temperature in the solution can be simulated with the liquid temperature of the cooling surface.

Preferably, in step (a), the difference between the temperature of the portion with the highest liquid temperature in the solution and the temperature of the portion with the lowest liquid temperature in the solution immediately before the heat of solidification is 2.5 ° C. or less The temperature is more preferably 2.3 ° C. or less, still more preferably 2.1 ° C. or less. Here, "the temperature difference immediately before the heat of solidification" means a temperature difference when the temperature difference becomes the largest between 1 second and 10 seconds before the heat of solidification.
Preferably, in the step (a), the temperature of the portion with the lowest liquid temperature in the solution is a solvent melting point of -5 ° C or less, more preferably a solvent melting point of -5 ° C or less and a solvent melting point of -20 ° C or more More preferably, the solvent melting point is -6 ° C or less and the solvent melting point -16 ° C or more. In addition, the solvent of a solvent melting point is a solvent of the solution of a biocompatible polymer.

  In step (b), the solution of the biocompatible polymer obtained in step (a) is frozen. The cooling temperature to be frozen in step (b) is not particularly limited, but it depends on the equipment to be cooled, but preferably from 3 ° C. to the temperature of the lowest temperature part in the solution. The temperature is 30 ° C. lower, more preferably 5 ° C. to 25 ° C. lower, still more preferably 10 ° C. to 20 ° C. lower.

  In step (c), the frozen biocompatible polymer obtained in step (b) is lyophilized. The lyophilization can be performed by a conventional method, for example, lyophilization can be performed by performing vacuum drying at a temperature lower than the melting point of the solvent and further performing vacuum drying at room temperature (20 ° C.).

(2) Biocompatible Polymer Porous Body According to the present invention, a biocompatible polymer porous body produced by the above-described method for producing a biocompatible polymer porous body is provided.
Furthermore, according to the present invention, a two-dimensional Fourier transform image obtained by applying a two-dimensional Fourier transform to a 1.5 mm square field of view of the cross-sectional structure of the biocompatible polymer porous body is compared with the two-dimensional When a line profile is created in which the luminance values around the x-axis coordinate are averaged for the 1/10 pixel width of the image pixel size from the bottom of the y-axis of the Fourier transform image and x is the line profile A biocompatible polymeric porous body is provided in which there is no peak at half of the maximum value.

  Here, "a peak does not exist when x of the line profile is half of the maximum value" preferably means that x of the line profile is half of the maximum value, where σ is the variance value of the base of the line profile. By the way, it means that the peak of 2.0σ or more is not detected.

  The method for obtaining the image of the cross-sectional structure of the biocompatible polymer porous body is not particularly limited. For example, it can be obtained by preparing a frozen section and preparing an HE (Hematoxylin Eosin))-stained specimen. A two-dimensional Fourier transform image is obtained by performing two-dimensional Fourier transform on a 1.5 mm square field of view in the obtained sample image. The two-dimensional Fourier transform can be performed, for example, using image analysis software ImageJ.

  In the obtained two-dimensional Fourier transform image, in the case where non-uniform directionality is confirmed in the biocompatible polymer porous body, a strong power spectrum is observed particularly in the axial direction. The presence or absence of the power spectrum in the axial direction can be defined by taking a line profile at the lower end of the y-axis of the two-dimensional Fourier transform image. Specifically, a line profile is created by averaging the luminance values around the x-axis coordinate and plotting on the y-axis from the lower end of the y-axis for a width of 1/10 of the image pixel size. The creation of the line profile can be performed, for example, using software “Gwyddion 2.31”. As described above, it is possible to define the presence or absence of a power spectrum by acquiring luminance values in the lateral direction of an image from end to end of the image and digitizing (line spectrum). The x-coordinate of the line profile is assumed that the left end of the two-dimensional Fourier transform image is zero, and the position advanced by one pixel to the right is 0.000001. That is, in the two-dimensional Fourier transform image, the position advanced 512 pixels from the left end has an x-coordinate of 0.000512 on the fine profile.

  The criterion for determining the presence or absence of the power spectrum is not particularly limited, but one example is 2 in the central portion (where x of the line profile is half of the maximum value), where σ is the variance value of the base of the obtained line profile. If no power spectrum is detected in the axial direction, and a peak of 2.0 σ or more is detected in the central portion (where x of the line profile is half the maximum value), It can be determined that there is a power spectrum in the axial direction.

  The “porous body” in the present invention is preferably a material having a plurality of “10 μm or more and 500 μm or less pores” in the inside of the main body when prepared as a 1 mm square material, and in the main body Although a material occupying 50% or more can be used, it is not particularly limited. In these materials, the internal pores described above may be in communication with each other, or some or all of the pores may be open on the surface of the material.

  The average pore size of the pores of the porous body can be obtained from observation of the cross-sectional structure of the porous body. First, the cross-sectional structure of the porous body is prepared as a sliced sample (for example, an HE-stained sample). Thereafter, among the walls formed of the polymer, the clear protrusion is connected to the closest protrusion and the holes are made clear. The divided individual void area thus obtained is measured, and then, the circle diameter when the area is converted to a circle is calculated. The obtained circle diameter can be used as the pore size, and an average value of 20 or more can be used as the average pore size.

  The biocompatible polymer porous body of the present invention can be used as a biocompatible polymer block (pulverized) as described below, but even when it is used as a porous body without being crushed, the cells are homogeneous. Can be Therefore, the biocompatible polymer porous body of the present invention is also useful as a culture carrier and a cell transplantation carrier.

(3) Biocompatibility Polymer The biocompatibility polymer used in the present invention is not particularly limited as to whether it is degraded in the living body as long as it has an affinity to the living body, but it is a biodegradable material It is preferred to be configured. Specific examples of non-biodegradable materials include polytetrafluoroethylene (PTFE), polyurethane, polypropylene, polyester, vinyl chloride, polycarbonate, acrylic, stainless steel, titanium, silicone, and MPC (2-methacryloyloxyethyl phosphorylcholine). At least one material selected from the group. Specific examples of biodegradable materials include polypeptides (for example, gelatin and the like described below), polylactic acid, polyglycolic acid, lactic acid / glycolic acid copolymer (PLGA), hyaluronic acid, glycosaminoglycan, proteoglycan, It is at least one material selected from the group of chondroitin, cellulose, agarose, carboxymethylcellulose, chitin and chitosan. Among the above, polypeptides are particularly preferred. These polymeric materials may be designed to improve cell adhesion, and specific methods include: “A cell adhesion substrate (fibronectin, vitronectin, laminin) to a substrate surface or a cell adhesion sequence (an amino acid one-letter code, RGD sequence, LDV sequence, REDV sequence, YIGSR sequence, PDSGR sequence, RYVVLPR sequence, LGTIPG sequence, RNIAEIIK DI sequence, IKVAV sequence, LRE sequence, DGEA sequence, and HAV sequence) coating with peptide ", "Amination of substrate surface, cationization", 3. Methods such as “plasma treatment of the substrate surface, hydrophilic treatment by corona discharge” may be used.

  The type of polypeptide is not particularly limited as long as it has biocompatibility, but for example, gelatin, collagen, elastin, fibronectin, ponectin, laminin, tenascin, fibrin, fibroin, entactin, thrombospondin, retronectin are preferable, Preferred are gelatin, collagen and atelocollagen. As gelatin for use in the present invention, natural gelatin or recombinant gelatin is preferable. More preferably, it is a recombinant gelatin. As used herein, natural gelatin means gelatin made from collagen of natural origin. The recombinant gelatin is described later in the specification.

The hydrophilicity value "1 / IOB" value of the biocompatible polymer used in the present invention is preferably 0 to 1.0. More preferably, it is 0 to 0.6, and more preferably 0 to 0.4. The IOB is an indicator of hydrophilicity / hydrophilicity based on an organic conceptual diagram representing the polarity / non-polarity of the organic compound proposed by Atsushi Fujita, and the details are described in, for example, "Pharmaceutical Bulletin", vol. 2, 2, pp .163-173 (1954), "Domain of Chemistry" vol. 11, 10, pp. 719-725 (1957), "Fragrance Journal", vol. 50, pp. 79-82 (1981), etc. There is. Briefly speaking, the source of all organic compounds is methane (CH ₄ ), and all the other compounds are regarded as derivatives of methane, and their carbon numbers, substituents, modified parts, rings, etc. are set to certain numerical values. The scores are added to obtain the organic value (OV) and the inorganic value (IV), and this value is plotted on the diagram with the organic value on the X axis and the inorganic value on the Y axis. It is something. The IOB in the organic conceptual diagram means the ratio of the inorganic value (IV) to the organic value (OV) in the organic conceptual diagram, that is, "inorganic value (IV) / organic value (OV)". For details of organic conceptual diagrams, refer to “New version of organic conceptual diagrams-basics and applications-” (Koda Yoshio et al., Sankyo Publishing, 2008). In the present specification, hydrophilicity is expressed by "1 / IOB" value which is the reciprocal of IOB. The smaller the “1 / IOB” value (closer to 0), the more hydrophilic the expression.

  By setting the “1 / IOB” value of the polymer used in the present invention to the above range, the hydrophilicity is high and the water absorbability is high, so it effectively acts on the retention of the nutritional component, and as a result, It is presumed to contribute to cell stabilization and survival in the cell three-dimensional structure (mosaic cell mass) of the invention.

When the bioaffinity polymer used in the present invention is a polypeptide, it is preferable that it is 0.3 or less, minus 9.0 or more in the hydrophilicity index indicated by Grand average of hydropathicity (GRAVY) value, More preferably, it is 0.0 or less and -7.0 or more. Grand average of hydropathicity (GRAVY) values can be obtained from Gasteiger E., Hoogland C., Gattiker A., Duvaud S., Wilkins MR, Appel RD, Bairoch A .; Protein Identification and Analysis Tools on the ExPASy Server; (In) John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press (2005). Pp. 571-607 and Gasteiger E., Gattiker A., Hoogland C., Ivanyi I., Appel RD, Bairoch A .; Nucleic Acids Res. 31: 3784-3788 (2003). "Can be obtained by the method of ExPASy: the proteomics server for in-depth protein knowledge and analysis.
By setting the GRAVY value of the polymer used in the present invention in the above-mentioned range, the hydrophilicity is high and the water absorptivity is high, so it effectively acts on the retention of the nutrient component, and as a result, the cell 3 of the present invention It is presumed to contribute to the stabilization and survival of cells in a three-dimensional structure (mosaic cell mass; mosaiced cell mass).

  The biocompatible polymer used in the present invention may be crosslinked or non-crosslinked, but is preferably crosslinked. Common crosslinking methods include thermal crosslinking, crosslinking with aldehydes (eg, formaldehyde, glutaraldehyde, etc.), crosslinking with condensing agents (carbodiimide, cyanamide, etc.), enzymatic crosslinking, photocrosslinking, ultraviolet crosslinking, hydrophobic interaction, Although hydrogen bonding, ionic interaction and the like are known, in the present invention, it is preferable to use a crosslinking method which does not use glutaraldehyde. The crosslinking method used in the present invention is more preferably thermal crosslinking, ultraviolet crosslinking or enzyme crosslinking, and particularly preferably thermal crosslinking.

  When performing crosslinking by an enzyme, the enzyme is not particularly limited as long as it has a crosslinking action between polymer materials, but preferably, crosslinking can be performed using transglutaminase and laccase, most preferably transglutaminase . A specific example of the protein which is enzymatically crosslinked by transglutaminase is not particularly limited as long as it is a protein having a lysine residue and a glutamine residue. The transglutaminase may be of mammalian origin or of microbial origin. Specifically, Ajinomoto's Activa series, mammalian transglutaminase released as a reagent, for example, Human-derived blood coagulation factors (Factor XIIIa, Haematologic Technologies, Inc., such as guinea pig liver-derived transglutaminase such as Oriental Yeast Co., Ltd., Upstate USA Inc., Biodesign International, goat-derived transglutaminase, rabbit-derived transglutaminase, etc. Company) and the like.

  The reaction temperature at the time of crosslinking (for example, thermal crosslinking) without using a crosslinking agent is not particularly limited as long as crosslinking is possible, but is preferably -100 ° C to 500 ° C, more preferably 0 ° C to 300 ° C. The temperature is more preferably 50 ° C. to 300 ° C., further preferably 100 ° C. to 250 ° C., and still more preferably 120 ° C. to 200 ° C.

  As the biocompatible polymer in the present invention, particularly preferably, recombinant gelatin can be used. The term "recombinant gelatin" refers to a polypeptide or protein-like substance having a gelatin-like amino acid sequence produced by genetic engineering techniques. The recombinant gelatin that can be used in the present invention is preferably one having a repeat of the sequence represented by Gly-X-Y characteristic of collagen (X and Y each independently represent any amino acid) (plural Gly-X-Y may be the same or different). Preferably, two or more sequences of cell adhesion signals are contained in one molecule. As the recombinant gelatin used in the present invention, a recombinant gelatin having an amino acid sequence derived from a partial amino acid sequence of collagen can be used, for example, those described in EP 1014176, US6992172, WO2004 / 85473, WO2008 / 103041 etc. Although it can be done, it is not limited to these. Preferred as the recombinant gelatin used in the present invention is a recombinant gelatin of the following aspect.

  The recombinant gelatin used in the present invention is excellent in non-infectiousness because it has excellent biocompatibility and is not of natural origin because of the natural performance of natural gelatin, and there is no concern such as bovine spongiform encephalopathy (BSE). In addition, since the recombinant gelatin used in the present invention is more uniform than the natural one and its sequence is determined, it is possible to design the strength and degradability with less blurring by the crosslinking etc. described later. is there.

  The molecular weight of the recombinant gelatin is preferably 2 kDa or more and 100 kDa or less. More preferably, they are 2.5 kDa or more and 95 kDa or less. More preferably, they are 5 kDa or more and 90 kDa or less. Most preferably, it is 10 kDa or more and 90 kDa or less.

  The recombinant gelatin has a repeat of the sequence shown by Gly-X-Y characteristic of collagen. Here, the plurality of Gly-X-Y may be the same or different. In Gly-X-Y, Gly represents glycine, and X and Y each represent any amino acid (preferably, any amino acid other than glycine). The GXY sequence characteristic of collagen is a highly specific partial structure as compared with other proteins in the amino acid composition and sequence of gelatin-collagen. In this part, glycine accounts for about one third of the whole, and in the amino acid sequence, one out of every three repeats. Glycine is the simplest amino acid, has less restriction on the arrangement of molecular chains, and greatly contributes to regeneration of the helical structure upon gelation. The amino acids represented by X and Y contain a large amount of imino acids (proline and oxyproline), and preferably occupy 10% to 45% of the total. Preferably, 80% or more, more preferably 95% or more, and most preferably 99% or more of the amino acids in the sequence have a GXY repeating structure.

  Among common polar amino acids, common gelatins have a charged one and an uncharged one at 1: 1. Here, polar amino acids specifically refer to cysteine, aspartic acid, glutamic acid, histidine, lysine, asparagine, glutamine, serine, threonine, tyrosine and arginine, and polar uncharged amino acids among these include cysteine, asparagine, glutamine, serine , Threonine, refers to tyrosine. In the recombinant peptide used in the present invention, the proportion of polar amino acids is 10 to 40%, preferably 20 to 30%, of all the constituent amino acids. And it is preferable that the proportion of uncharged amino acids in the polar amino acids is 5% or more and less than 20%, preferably less than 10%. Furthermore, it is preferable not to include any one amino acid, preferably two or more amino acids of serine, threonine, asparagine, tyrosine and cysteine on the sequence.

  Generally, in polypeptides, the minimum amino acid sequence that works as a cell adhesion signal is known (eg, “Pathophysiology” Vol. 9, No. 7 (1990) page 527) published by Nagai Publishing Co., Ltd. The recombinant gelatin used in the present invention preferably has two or more of these cell adhesion signals in one molecule. Specific sequences include RGD sequence, LDV sequence, REDV sequence, YIGSR sequence, PDSGR sequence, RYVVLPR sequence, LGTIPG sequence, RNIAEIIKDI sequence, which is expressed by single-letter amino acid notation in that there are many types of cells to adhere. The sequences of IKVAV, LRE, DGEA and HAV are preferable, more preferably RGD, YIGSR, PDSGR, LGTIPG, IKVAV and HAV, particularly preferably RGD. Among the RGD sequences, preferred are ERGD sequences. By using a recombinant gelatin having a cell adhesion signal, it is possible to improve the amount of substrate production of cells. For example, in the case of cartilage differentiation using mesenchymal stem cells as cells, the production of glycosaminoglycan (GAG) can be improved.

  As arrangement | positioning of the RGD sequence in the recombinant gelatin used by this invention, it is preferable that the amino acid number between RGD is not uniform between 0-100, Preferably between 25-60.

  The content of the minimum amino acid sequence is preferably 3 to 50, more preferably 4 to 30, and particularly preferably 5 to 20 in one molecule of protein from the viewpoint of cell adhesion and proliferation. Most preferably, it is twelve.

  In the recombinant gelatin used in the present invention, the ratio of the RGD motif to the total number of amino acids is preferably at least 0.4%, and when the recombinant gelatin contains 350 or more amino acids, each stretch of 350 amino acids is at least one RGD It is preferred to include a motif. The ratio of the RGD motif to the total number of amino acids is more preferably at least 0.6%, more preferably at least 0.8%, still more preferably at least 1.0%, further preferably at least 1.2%. Most preferably at least 1.5%. The number of RGD motifs in the recombinant peptide is preferably at least 4, more preferably 6, more preferably 8, still more preferably 12 to 16, per 250 amino acids. A percentage of 0.4% of the RGD motif corresponds to at least one RGD sequence per 250 amino acids. Because the number of RGD motifs is an integer, gelatin consisting of 251 amino acids must contain at least two RGD sequences in order to meet the 0.4% feature. Preferably, the recombinant gelatin of the present invention comprises at least 2 RGD sequences per 250 amino acids, more preferably at least 3 RGD sequences per 250 amino acids, more preferably at least 4 per 250 amino acids. Contains RGD sequence. A further embodiment of the recombinant gelatin of the present invention comprises at least 4 RGD motifs, preferably 6, more preferably 8 and even more preferably 12 or more and 16 or less.

  In addition, the recombinant gelatin may be partially hydrolyzed.

Preferably, the recombinant gelatin used in the present invention is one represented by the formula: A-[(Gly-X-Y) _n ] _m -B. The n X's each independently represent any of the amino acids, and the n Y's each independently represent any of the amino acids. Preferably it is 2-10, preferably 3-5 as m. n is preferably 3 to 100, more preferably 15 to 70, and most preferably 50 to 65. A represents any amino acid or amino acid sequence, B represents any amino acid or amino acid sequence, n Xs each independently represent any of amino acids, and n Ys each independently represent any of amino acids Show.

More preferably, the recombinant gelatin used in the present invention is represented by the formula: Gly-Ala-Pro-[(Gly-X-Y) ₆₃ ] ₃ -Gly (wherein each of 63 Xs independently represents any amino acid) And each of the 63 Ys independently represents any amino acid, and the 63 Gly-X-Ys may be the same or different.
It is preferred that a plurality of sequence units of naturally occurring collagen be bound to the repeating unit. The naturally occurring collagen referred to here may be any naturally occurring collagen, but preferably it is type I, type II, type III, type IV and type V. More preferably, they are types I, II and III. According to another form, the collagen is preferably human, bovine, porcine, murine, rat. More preferably, it is a human.

The isoelectric point of the recombinant gelatin used in the present invention is preferably 5 to 10, more preferably 6 to 10, and still more preferably 7 to 9.5.
Preferably, the recombinant gelatin is not deaminated.
Preferably, the recombinant gelatin does not have telopentide.
Preferably, the recombinant gelatin is a substantially pure collagen material prepared with nucleic acid encoding natural collagen.

As the recombinant gelatin used in the present invention, preferably
(A) a protein having the amino acid sequence set forth in SEQ ID NO: 1;
(B) a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1 and having biocompatibility; or (c) described in SEQ ID NO: 1 A protein having an amino acid sequence having a homology of 80% or more (more preferably 90% or more, more preferably 95% or more) with the amino acid sequence of
It is either.

More preferably, the recombinant gelatin used in the present invention is
(A) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1;
(B) An amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1, and a protein having biocompatibility; or (c) SEQ ID NO: 1 A protein consisting of an amino acid sequence having 80% or more (more preferably 90% or more, more preferably 95% or more) homology with the amino acid sequence, and a protein having biocompatibility;
It is either.

  The "one or several" in the "amino acid sequence in which one or several amino acids are deleted, substituted or added" is preferably 1 to 20, more preferably 1 to 10, still more preferably 1 to 5. It means one, particularly preferably 1 to 3.

  The recombinant gelatin used in the present invention can be produced by gene recombination technology known to those skilled in the art, and can be produced, for example, according to the method described in EP1014176A2, US6992172, WO2004-85473, WO2008 / 103041 and the like. Specifically, a gene encoding an amino acid sequence of a predetermined recombinant gelatin is obtained and incorporated into an expression vector to prepare a recombinant expression vector, which is introduced into an appropriate host to prepare a transformant. . Since the recombinant gelatin is produced by culturing the obtained transformant in an appropriate medium, the recombinant gelatin used in the present invention can be prepared by recovering the recombinant gelatin produced from the culture. .

(4) Biocompatible Polymer Block The method for producing the biocompatible polymer block (mass containing the biocompatible polymer) is not particularly limited. For example, a porous body of the biocompatible polymer is milled. The biocompatible polymer block can be obtained by grinding using a New Power Mill or the like.

The shape of the biocompatible polymer block in the present invention is not particularly limited.
The size of one biocompatible polymer block in the present invention is preferably 20 μm or more and 200 μm or less. More preferably, they are 20 micrometers or more and 120 micrometers or less, More preferably, they are 50 micrometers or more and 120 micrometers or less. By making the size of one biocompatible polymer block within the above range, better cell viability can be achieved. The size of one biocompatible polymer block does not mean that the average value of the sizes of a plurality of biocompatible polymer blocks is in the above range, and a plurality of biocompatible polymers are used. It means the size of each biocompatible polymer block obtained by sieving the block.

  As described later in the present specification, the cell structure of the present invention can be produced by mixing the biocompatible polymer block of the present invention and at least one type of cell. That is, the biocompatible polymer block of the present invention is useful as a reagent for producing the cell structure of the present invention.

(5) Cell As the cell used in the present invention, any cell can be used as long as it can perform cell transplantation, which is the object of the cell structure of the present invention, and the type thereof is not particularly limited. Moreover, cells of one type may be used, or plural types of cells may be used in combination. The cells to be used are preferably animal cells, more preferably vertebrate-derived cells, particularly preferably human-derived cells. The type of vertebrate-derived cells (in particular, human-derived cells) may be any of universal cells, somatic stem cells, progenitor cells, or mature cells. As pluripotent cells, for example, ES cells, GS cells, or iPS cells can be used. As somatic stem cells, for example, mesenchymal stem cells (MSCs), hematopoietic stem cells, amniocytes, cord blood cells, bone marrow-derived cells, cardiac muscle stem cells, adipose-derived stem cells, or neural stem cells can be used. As precursor cells and mature cells, for example, skin, dermis, epidermis, muscle, myocardium, nerve, bone, cartilage, endothelium, brain, epithelium, heart, kidney, liver, pancreas, spleen, oral cavity, cornea, bone marrow, umbilical cord Cells derived from blood, amniotic membrane or hair can be used. Examples of human-derived cells include ES cells, iPS cells, MSCs, chondrocytes, osteoblasts, osteoblast precursor cells, mesenchymal cells, myoblasts, cardiomyocytes, cardiac myoblasts, neurons, hepatocytes, Beta cells, fibroblasts, corneal endothelial cells, vascular endothelial cells, corneal epithelial cells, amniocytes, cord blood cells, bone marrow derived cells, or hematopoietic stem cells can be used. Also, the origin of the cells may be either autologous cells or allogeneic cells.

  For example, in heart diseases such as severe heart failure and severe myocardial infarction, cardiomyocytes, smooth muscle cells, fibroblasts, skeletal muscle-derived cells (especially satellite cells), bone marrow cells (especially myocardial-like cells) isolated from autologous and allogeneic Bone marrow cells (differentiated bone marrow cells) and the like can be suitably used. Furthermore, transplanted cells can be appropriately selected in other organs. For example, neural progenitor cells to brain ischemia / infarction site, transplantation of cells capable of differentiating to nerve cells, vascular endothelial cells to myocardial infarction site / skeletal muscle ischemic site or cells capable of differentiating to vascular endothelial cells Transplantation etc. can be mentioned.

  In addition, cells used for cell transplantation for diabetic organ damage can be mentioned. For example, cells for cell transplantation therapy which are variously studied for diseases such as kidney, pancreas, peripheral nerve, eye, limb circulation disorder and the like can be mentioned. That is, attempts have been made to transplant insulin-secreting cells to a pancreas having a reduced ability to secrete insulin, and transplantation of bone marrow-derived cells for impaired circulation in limbs, etc., and such cells can be used.

  In the present invention, cells of vasculature can also be used. As used herein, the cells of the vasculature mean cells involved in angiogenesis, cells constituting blood vessels and blood, and precursor cells capable of differentiating into the cells, somatic stem cells. Here, cells of the vascular system do not spontaneously differentiate into cells constituting blood vessels and blood, such as pluripotent cells such as ES cells, GS cells, or iPS cells, and mesenchymal stem cells (MSCs). Things are not included. The cells of the vascular system are preferably cells that constitute a blood vessel. In vertebrate-derived cells (in particular, human-derived cells), vascular endothelial cells and vascular smooth muscle cells can be suitably exemplified as cells that constitute blood vessels. Vascular endothelial cells include both venous and arterial endothelial cells. Vascular endothelial progenitor cells can be used as progenitor cells for vascular endothelial cells. Preferred are vascular endothelial cells and vascular endothelial precursor cells. Blood cells can be used as cells constituting blood, and white blood cells such as lymphocytes and neutrophils, monocytes, and hematopoietic stem cells which are stem cells thereof can be used.

  As used herein, non-vascular cells mean cells other than the above-mentioned vasculature. For example, ES cells, iPS cells, mesenchymal stem cells (MSCs), cardiac muscle stem cells, cardiac muscle cells, fibroblasts, myoblasts, chondrocytes, myoblasts, hepatocytes or neurons can be used. Preferably, mesenchymal stem cells (MSCs), chondrocytes, myoblasts, cardiac stem cells, cardiomyocytes, hepatocytes or iPS cells can be used. More preferably, mesenchymal stem cells (MSCs), cardiac stem cells, cardiac myocytes or myoblasts.

(6) Cell Structure The cell structure of the present invention comprises the above-described biocompatibility polymer block of the present invention and at least one type of cell, and a plurality of bioaffinities in the interstices of a plurality of cells It is a cell structure in which a polymer block is arranged. The cell construct of the present invention can be used for cell transplantation.

  In the present invention, using the above-described biocompatibility polymer block and the above-described cells, cells are arranged in a three-dimensional manner in a mosaic manner between the plurality of cells by arranging the plurality of polymer blocks in a three-dimensional manner. It is possible to have a thickness suitable for transplantation, and by arranging the biocompatible polymer block and the cells in a three-dimensional manner in a mosaic manner, the cells are uniformly present in the structure 3 A dimensional structure is formed, which enables nutrient delivery from the outside into the interior of the cell three-dimensional structure.

  In the cell structure of the present invention, a plurality of polymer blocks are disposed in a gap between a plurality of cells, wherein “the gap between cells” is closed by cells that are configured. It does not have to be a space, as long as it is sandwiched by cells. In addition, it is not necessary to have a gap between all the cells, and there may be a place where the cells are in contact with each other. There is no particular limitation on the distance between the cells via the polymer block, that is, when selecting a cell and a cell located at the shortest distance from the cell, the size of the polymer block is not particularly limited. And the preferred distance is also in the preferred size range of the polymer block.

  In addition, although the polymer block according to the present invention is configured to be sandwiched by cells, there is no need for cells to be present between all the polymer blocks, and there may be portions where polymer blocks are in contact with each other. . There is no particular limitation on the distance between the polymer blocks through the cells, ie, when the polymer block and the polymer block located at the shortest distance from the polymer block are selected, there is no particular limitation. It is preferable that the size of a cell mass when 1 to several pieces gather is, for example, 10 μm or more and 1000 μm or less, preferably 10 μm or more and 100 μm or less, and more preferably 10 μm or more and 50 μm or less.

  In the present specification, although the expression “uniformly exists” such as “cell three-dimensional structure in which cells uniformly exist in the structure” is used, it means complete uniformity. Instead, it means that nutrient delivery from the outside to the inside of the three-dimensional cell structure is enabled, which is the effect of the present invention.

  The thickness or diameter of the cell structure of the present invention can be a desired thickness, but the lower limit is preferably 215 μm or more, more preferably 400 μm or more, and most preferably 730 μm or more. The upper limit of the thickness or diameter is not particularly limited, but as a general range of use, 3 cm or less is preferable, 2 cm or less is more preferable, and 1 cm or less is more preferable. The thickness or the diameter of the cell structure is preferably in the range of 400 μm to 3 cm, more preferably 500 μm to 2 cm, and still more preferably 720 μm to 1 cm.

  In the cell structure of the present invention, preferably, the region consisting of the polymer block and the region consisting of the cells are arranged in a mosaic. In addition, "the thickness or diameter of a cell structure" in this specification shall show the following thing. When a certain point A in the cell structure is selected, the length of a line segment that divides the cell structure so as to be the shortest distance from the outside of the cell structure within the straight line passing that point A is a line Let it be minute A. The point A at which the line segment A is longest in the cell structure is selected, and the length of the line segment A at that time is taken as the "thickness or diameter of the cell structure".

  When the cell structure of the present invention is used as a cell structure before fusion in the method for producing a cell structure of the present invention described later, or a cell structure before addition of a second polymer block, The thickness or diameter of the cell structure is preferably 10 μm to 1 cm, more preferably 10 μm to 2000 μm, still more preferably 15 μm to 1500 μm, and most preferably 20 μm to 1300 μm.

  In the cell structure of the present invention, the ratio of cells to polymer blocks is not particularly limited, but the ratio of polymer blocks per cell is preferably 0.0000001 μg or more and 1 μg or less, and more preferably 0. And more preferably 0.00001 μg or more and 0.01 μg or less, and most preferably 0.00002 μg or more and 0.006 μg or less. With this range, cells can be more uniformly present. In addition, by setting the lower limit to the above range, the effect of the cell can be exhibited when used for the above-mentioned application, and by setting the upper limit to the above range, the component in the polymer block optionally present can be It can be supplied. Here, the components in the polymer block are not particularly limited, but include the components contained in the culture medium described later.

  In addition, the cell structure of the present invention may contain an angiogenic factor. Here, as the angiogenic factor, basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and the like can be suitably mentioned. The method for producing a cell structure containing an angiogenic factor is not particularly limited, and can be produced, for example, by using a polymer block impregnated with an angiogenic factor. From the viewpoint of promoting angiogenesis, the cell structure of the present invention preferably contains an angiogenic factor.

  As the cell structure of the present invention, those containing cells of non-vasculature can also be suitably used. Moreover, cells in which the cells constituting the cell structure of the present invention are only cells of non-vasculature can also be suitably used. The cell structure of the present invention containing only non-vascular cells as cells enables blood vessels to be formed at the transplantation site after transplantation. Also, in the case where the cells constituting the cell structure of the present invention are two or more types, and include both non-vascular system cells and vascular system cells, it may be composed of only non-vascular system cells and In comparison, more blood vessels can be formed, which is preferable.

  Furthermore, when there are two or more types of cells constituting the cell structure of the present invention, and the area of the cells of the vascular system at the center of the cell structure is larger than the area of the cells of the vascular system at the periphery. Further, it becomes possible to form blood vessels, which is more preferable. Here, if the area of the cells of the vascular system at the center of the cell structure has a larger area than the area of the cells of the vascular system at the periphery, specifically, an arbitrary 2 μm thin-sectioned sample was prepared Sometimes we say that there is a specimen with the area that is above. Here, the central portion of the cell structure means an area at a distance of 80% from the center to the surface of the cell structure, and the peripheral portion of the cell structure is 80 The area from the location of% to the surface of the structure. The central part of the cell structure is defined as follows.

  In any cross section passing through the center of the cell structure, the center of the circle of radius X is moved along the extension of the cross section, and the area of the moved circle excluding the overlapping portion of the cross section is the cross section of the cross section. Find a radius X that is 64% of the area. The center of the circle of radius X is moved around, and the portion excluding the overlapping portion of the moved circle and the cross section is taken as the central portion of the cell structure. At this time, the cross section where the cross-sectional area is the largest is most preferable. In the cross-section where the cross-sectional area is the largest, the center of the cell structure moves the center of the circle of radius Y around the circumference of the cross-section and removes the overlapping part of the moved circle and the cross-section The radius Y which is decided to one point is determined. The center of the circle of radius Y is moved around, and one point excluding the overlapping portion of the moved circle and the cross section is made the center of the cell structure. When it does not decide to one point and it becomes a line segment, or when there are a plurality of line segments, for each of the line segments, the point at which the length is bisected is the center.

  Specifically, it is preferable to have a region in which the proportion of cells in the central vasculature is 60% to 100% of the total area of the cells in the vasculature, and more preferably 65% to 100%, 80 % To 100% is more preferable, and 90% to 100% is further preferable. In addition, here, having a region in which the percentage of vascular cells in the central part is 60% to 100% with respect to the total area of vascular cells is specifically an arbitrary 2 μm thin section When a sample is prepared, it is said that a sample having the area of the proportion is present. By setting it as the said range, angiogenesis can be promoted more.

  In addition, as for the proportion of cells in the central vasculature, for example, when a thin-sectioned sample is prepared, the cells in the vasculature to be measured are stained, and the color density of the central portion is obtained using the image processing software ImageJ ( Average value of intensity) is calculated, area of central part × intensity is calculated, average value of intensity (intensity) of the whole color is further calculated, area × intensity of the whole is calculated, area × intensity of the whole It can also be calculated by determining the ratio of area × intensity at the center. Here, as a method of staining cells of the vascular system, known staining methods can be appropriately used. For example, when using human vascular endothelial precursor cells (hECFC) as the cells, a CD31 antibody is used. can do.

In addition, it is preferable to have a region in which the cell density of the vascular system in the central part of the cell structure is 1.0 × 10 ⁻⁴ cells / μm ³ or more, and the cell density is the entire cell center. Is more preferred. Here, having an area of 1.0 × 10 ⁻⁴ cells / μm ³ or more specifically refers to a sample having an area of the density when an arbitrary 2 μm sliced sample is prepared. Say that exists. The cell density is more preferably ^{1.0 × 10 -4 ~1.0 × 10 -3} cells / μm 3, more preferably ^{1.0 × 10 -4 ~2.0 × 10 -4} cells / μm 3 More preferably, it is 1.1 × 10 ^{−4 to} 1.8 × 10 ⁻⁴ cells / μm ³ , more preferably 1.4 × 10 ^{−4 to} 1.8 × 10 ⁴ cells / μm ³ . By setting it as the said range, angiogenesis can be promoted more.

  Here, the cell density of the central vasculature can be determined by actually counting the number of cells of the sliced sample and dividing the number of cells by volume. The central part here is defined as follows. The thickness of the sliced sample is cut in the direction perpendicular to the central portion described above. The cell density can be determined, for example, by superimposing the sliced sample in which the cells of the blood vessel system to be measured are stained with the sliced sample in which the cell nucleus is stained, and counting the number of cell nuclei overlapped. The number of cells in the vasculature can be calculated, and the volume can be obtained by calculating the area of the central part using ImageJ and multiplying the thickness of the sliced sample.

  The cell structure of the present invention also includes those formed into blood vessels using the cell structure of the present invention, which comprises two or more types of cells, and includes both non-vascular cells and cells of vascular system. . Also, here, the preferable range of the “cell structure of the present invention in which the cells are two or more types and includes both non-vascular cells and vascular cells” is the same as above. As a method of constructing a blood vessel, for example, there is a method in which a cell sheet in which vascular cells are mixed is attached to a gel material in which a blood vessel portion is tunneled, and culture is performed while flowing a culture solution in a tunnel. In addition, blood vessels can also be constructed by sandwiching vascular cells between cell sheets.

(7) Method of Producing Cell Structure The cell structure of the present invention can be produced by mixing the biocompatible polymer block of the present invention with at least one type of cell. More specifically, the cell structure of the present invention can be produced by alternately arranging a biocompatible polymer block (a mass consisting of a biocompatible polymer) and cells. Although the production method is not particularly limited, it is preferably a method of seeding cells after forming a polymer block. Specifically, the cell structure of the present invention can be produced by incubating a mixture of a biocompatible polymer block and a cell-containing culture solution. For example, cells and a biocompatible polymer block prepared in advance are disposed in a mosaic shape in a container and in a liquid held in the container. As means of arrangement, it is preferable to promote or control the formation of a mosaic-like array of cells and a biocompatible substrate by using natural aggregation, natural fall, centrifugation, or agitation.

  The container to be used is preferably a container made of a low cell adhesion material or a non-cell adhesion material, more preferably a container made of polystyrene, polypropylene, polyethylene, glass, polycarbonate, polyethylene terephthalate. The shape of the bottom of the container is preferably flat bottom, U-shaped or V-shaped.

The mosaic cell structure obtained by the above method is, for example,
(A) fusing separately prepared mosaic-like cell masses, or (b) increasing the volume under differentiation medium or growth medium,
Cell structures of a desired size can be produced by methods such as, for example. The method of fusion and the method of volume up are not particularly limited.

  For example, in the step of incubating the mixture of the biocompatible polymer block and the cell-containing culture solution, the cell structure can be volume-increased by replacing the medium with a differentiation medium or a growth medium. Preferably, in the step of incubating the mixture of the biocompatible polymer block and the cell-containing culture medium, a cell structure of a desired size, which is a cell structure of a desired size, by further adding the biocompatible polymer block. Cell structures in which cells are uniformly present in the body can be produced.

  Specifically, the method of fusing the mosaic-like cell masses separately prepared includes a plurality of biocompatible polymer blocks and a plurality of cells, and a plurality of cells formed by the plurality of cells A method for producing a cell structure, which comprises the step of fusing a plurality of cell structures in which one or a plurality of the biocompatibility polymer blocks are disposed in part or all of individual gaps.

  "Biocompatible polymer block (type, size, etc.)", "cells", "gap between cells", "cell structure obtained (size, etc.)" according to the method for producing a cell structure of the present invention Preferred ranges of “ratio of cell to polymer block” and the like are the same as those described above in the present specification.

  The thickness or diameter of each cell structure before the fusion is preferably 10 μm to 1 cm, and the thickness or diameter after the fusion is preferably 400 μm to 3 cm. Here, the thickness or diameter of each cell structure before the fusion is more preferably 10 μm or more and 2000 μm or less, still more preferably 15 μm or more and 1500 μm or less, and most preferably 20 μm or more and 1300 μm or less. More preferably, it is 500 micrometers or more and 2 cm or less, and still more preferably 720 micrometers or more and 1 cm or less.

  More specifically, the method for producing a cell structure having a desired size by further adding the above-described biocompatibility polymer block includes a plurality of first biocompatibility polymer blocks and a plurality of plurality of biocompatibility polymer blocks. Furthermore, in a cell structure including one or more of the biocompatibility polymer blocks disposed in part or all of a plurality of gaps formed by the plurality of cells, It is a method for producing a cell structure, which comprises the steps of adding and incubating a second biocompatible polymer block. Here, “biocompatible polymer block (type, size, etc.)”, “cell”, “gap between cells”, “cell structure obtained (size, etc.)”, “cell and polymer block Preferred ranges such as “ratio” are as described above in the present specification.

  Here, the cell structures to be fused are preferably placed at a distance of 0 to 50 μm, more preferably 0 to 20 μm, and still more preferably 0 to 5 μm. When fusing cell structures with each other, it is considered that cells or cell-produced substrates play a role as an adhesive by cell growth and extension, and they are considered to be joined. Becomes easy.

  The thickness or diameter of the cell structure obtained by the method for producing a cell structure of the present invention is preferably 400 μm or more and 3 cm or less, more preferably 500 μm or more and 2 cm or less, and still more preferably 720 μm or more and 1 cm or less.

  Furthermore, when adding and incubating a second biocompatible polymer block to the cell structure, the pace of adding the second biocompatible polymer block is matched to the growth rate of the cells used, It is preferable to select suitably. Specifically, when the second pace of addition of the second biocompatible polymer block is accelerated, the cells move to the outside of the cell structure, the uniformity of the cells decreases, and the pace of addition decreases. Since there are many places where the proportion is high and the cell uniformity is low, the growth rate of the cells to be used is considered and selected.

As a manufacturing method of a cell structure in the case of including both non-vascular cells and vascular cells, for example, the following manufacturing methods (a) to (c) can be suitably mentioned.
(A) is a production method comprising the step of forming cell structures using non-vasculature cells by the method described above, and then adding vasculocytes and a biocompatible polymer block. Here, “the step of adding the cells of the vascular system and the high biocompatibility molecular block” refers to the method of fusing the prepared mosaic-like cell masses with each other and the method of increasing the volume under differentiation medium or growth medium as described above. , Including all. According to this method, (i) the area of non-vasculature cells is larger in the central part of the cell structure as compared to the cells of the vasculature, and in the peripheral part, the vasculature is more compared to the non-vascular cells. A cell structure with a large area of cells, (ii) a cell structure in which the area of central non-vascular cells is larger than the area of peripheral non-vascular cells, (iii) cells It is possible to produce a cell structure in which the area of the central vasculature cells of the construct is less than the area of the peripheral vasculature cells.
(B) is a production method comprising the steps of forming cell structures by using the cells of the vasculature by the method described above, and then adding non-vasculature cells and a biocompatible polymer block. Here, "the step of adding non-vascular cells and a biocompatible polymer block" refers to the method of fusing the prepared mosaic-like cell masses with each other and the volume increase under differentiation medium or growth medium as described above. Methods, including all. According to this method, (i) the area of the cells of the duct system is larger in the central part of the cell structure compared to the cells of the non vascular system, and in the peripheral part, the non vascular system is compared with the cells of the vascular system. A cell structure with a large area of cells, (ii) a cell structure in which the area of cells in the central vasculature of the cell structure is larger than the area of cells in the peripheral vasculature, (iii) the cell structure It is possible to produce a cell structure in which the area of non-vascular cells in the central part is smaller than the area of non-vascular cells in the peripheral part.
(C) is a manufacturing method in which cells of non-vascular system and cells of vascular system are substantially simultaneously used to form a cell structure by the method described above. In this method, it is possible to produce a cell structure in which any of non-vasculature cells and cells of vasculature do not become largely unevenly distributed at any part of the cell structure.

From the viewpoint of forming blood vessels at the transplantation site after transplantation, the area of the cells of the vascular system is larger at the center of the cell structure compared to the cells of the non-vascular system, and compared to the cells of the vascular system at the periphery. Preferably, the cell structure is a cell structure in which the area of non-vascular cells is large, and the area of cells in the central vasculature is larger than the area of cells in the peripheral vasculature, Angiogenesis can be further promoted by using a cell structure. Furthermore, in the cell structure, the larger the number of cells present in the center, the more the blood vessel formation can be promoted.
For the same reason, it is preferable to have a production method comprising the steps of forming cell structures with cells of vasculature and then adding non-vasculature cells and a biocompatible polymer block. And, it is more preferable to increase the number of cells in the vascular system.

(8) Use of Cell Structure The cell structure of the present invention can be used, for example, for the purpose of cell transplantation at a disease site such as a heart failure such as severe heart failure or severe myocardial infarction, or cerebral ischemia or cerebral infarction. In addition, it can also be used for diseases such as diabetic kidney, pancreas, peripheral nerve, eye, limb circulation disorder and the like. As an implantation method, an incision, an injection, an endoscope and the like can be used. The cell construct of the present invention can reduce the size of the construct, unlike cell transplants such as cell sheets, thereby enabling a minimally invasive transplantation method such as implantation by injection.

  Also, according to the present invention, a cell transplantation method is provided. The cell transplantation method of the present invention is characterized by using the above-mentioned cell structure of the present invention. The preferred range of the cell construct is the same as described above.

  The present invention will be described more specifically by the following examples, but the present invention is not limited by the examples.

[Materials and container]
[Recombinant peptide (recombinant gelatin)]
The CBE described below was prepared as a recombinant peptide (recombinant gelatin) (described in WO2008-103041).
CBE3
Molecular weight: 51.6 kD
Structure: GAP [(GXY) ₆₃ ] ₃ G
Amino acid number: 571 RGD sequence: 12 imino acid content: 33%
Almost 100% of the amino acids are GXY repeating structures. The amino acid sequence of CBE3 does not include serine, threonine, asparagine, tyrosine and cysteine. CBE3 has the ERGD sequence.
Isoelectric point: 9.34, GRAVY value: -0.682, 1 / IOB value: 0. 323

Amino acid sequence (SEQ ID NO: 1 in the sequence listing) (same as SEQ ID NO: 3 of WO2008 / 103041. However, X at the end is corrected to "P")
GAP (GAPGLQ GAPGLQ GMP GERGAAGLPGPKGERGDAGPKGAD GAP GAP GAP GAP GAP ERG AGAGPGPG ERG ERG GP KG KD KD G A A A A A G GAP GAP GAP GAP GAP GAP GAP GAP GAP GAP GAP GAP GAP GAP GAP GAP GAP GAP GAP GAP GAP GAP GAP GAP GAP GAP GAP

[PTFE thick and cylindrical container]
A polytetrafluoroethylene (PTFE) cylindrical cup-shaped container having a bottom thickness of 3 mm, a diameter of 51 mm, a side thickness of 8 mm and a height of 25 mm was prepared. When the cylindrical cup has a curved side, the side is closed with 8 mm of PTFE, and the bottom surface (the circular shape of the flat plate) is also closed with 3 mm of PTFE. On the other hand, the upper surface has an open shape. Thus, the inner diameter of the cylindrical cup is 43 mm. Hereinafter, this container is referred to as a PTFE thick / cylindrical container.

[Aluminum glass plate / cylindrical container]
An aluminum cylindrical cup-shaped container having a thickness of 1 mm and a diameter of 47 mm was prepared. When the cylindrical cup is a curved side, the side is closed with 1 mm of aluminum, and the bottom surface (the circular shape of the flat plate) is also closed with 1 mm of aluminum. On the other hand, the upper surface has an open shape. In addition, 1 mm thick Teflon (registered trademark) is uniformly spread only inside the side surface, and as a result, the inner diameter of the cylindrical cup is 45 mm. In addition, a 2.2 mm glass plate is bonded to the bottom of the container in addition to aluminum. Hereinafter, this container is referred to as an aluminum glass / cylindrical container.

Example 1
[Freezing process with small temperature difference, and drying process]
The CBE3 aqueous solution was poured into a PTFE thick / cylindrical container, an aluminum glass plate / cylindrical container, and the CBE3 aqueous solution was cooled from the bottom using a cooling shelf in a vacuum freeze dryer (TF5-85ATNNN: Takara Seisakusho).
The combination of the container, the final concentration of the CBE3 aqueous solution, the amount of liquid, and the setting of the shelf plate temperature at this time was prepared as described below.

"A" PTFE thick cylindrical container, final concentration 4% by mass of CBE3 aqueous solution, 4 mL aqueous solution volume. Set the shelf temperature to -10 ° C, cool at -10 ° C for 1 hour, then at -20 ° C for 2 hours, then at -40 ° C for 3 hours and finally at -50 ° C for 1 hour. The The frozen product is then vacuum dried at -20 ° C for 24 hours after returning the shelf temperature to -20 ° C, and after 24 hours, the shelf temperature is raised to 20 ° C while vacuum drying is continued. After vacuum drying was further performed at 20 ° C. for 48 hours until the degree of vacuum was sufficiently lowered (1.9 × 10 ⁵ Pa), it was removed from the vacuum lyophilizer. A porous body was thereby obtained.
"A" Aluminum, glass plate, cylindrical container, final concentration 4% by mass of CBE3 aqueous solution, aqueous solution volume 4 mL. Set the shelf temperature to -10 ° C, cool at -10 ° C for 1 hour, then at -20 ° C for 2 hours, then at -40 ° C for 3 hours and finally at -50 ° C for 1 hour. The The frozen product is then vacuum dried at -20 ° C for 24 hours after returning the shelf temperature to -20 ° C, and after 24 hours, the shelf temperature is raised to 20 ° C while vacuum drying is continued. After vacuum drying was further performed at 20 ° C. for 48 hours until the degree of vacuum was sufficiently lowered (1.9 × 10 ⁵ Pa), it was removed from the vacuum lyophilizer. A porous body was thereby obtained.
"C" PTFE thick cylindrical container, final concentration 4% by mass of CBE3 aqueous solution, 10 mL aqueous solution volume. Set the shelf temperature to -10 ° C, cool at -10 ° C for 1 hour, then at -20 ° C for 2 hours, then at -40 ° C for 3 hours and finally at -50 ° C for 1 hour. The The frozen product is then vacuum dried at -20 ° C for 24 hours after returning the shelf temperature to -20 ° C, and after 24 hours, the shelf temperature is raised to 20 ° C while vacuum drying is continued. After vacuum drying was further performed at 20 ° C. for 48 hours until the degree of vacuum was sufficiently lowered (1.9 × 10 ⁵ Pa), it was removed from the vacuum lyophilizer. A porous body was thereby obtained.

Comparative Example 1
[Freezing process with large temperature difference, and drying process]
4 mL of CBE 3 aqueous solution was poured into an aluminum glass plate / cylindrical container, and the CBE 3 aqueous solution was cooled from the bottom using a cooling shelf in a freezer. The combination of the container, the final concentration of the CBE3 aqueous solution, the amount of liquid, and the setting of the shelf plate temperature at this time was prepared as described below. Moreover, freezing was implemented by installing the container containing CBE3 aqueous solution to the place which shelf plate temperature fully cooled beforehand until it became preset temperature.

"D" Aluminum glass plate / cylindrical container, final concentration 4% by mass of CBE3 aqueous solution, 4 mL aqueous solution volume. The shelf temperature was set by placing an aluminum glass plate / container with CBE3 aqueous solution in a place where it had been cooled to -60 ° C in advance, and freezing was performed while maintaining the shelf temperature at -60 ° C. . The frozen product was then freeze-dried to obtain a porous body.
"A" Aluminum glass plate / cylindrical container, final concentration of CBE3 aqueous solution 7.5% by mass, amount of aqueous solution 4 mL. The shelf temperature was set by placing an aluminum glass plate / container with CBE3 aqueous solution in a place where it had been cooled to -60 ° C in advance, and freezing was performed while maintaining the shelf temperature at -60 ° C. . The frozen product was then freeze-dried to obtain a porous body.
"Ca" Aluminum glass plate / cylindrical container, final concentration 4% by mass of CBE3 aqueous solution, 4 mL aqueous solution volume. The shelf temperature was set by placing an aluminum glass plate / container with CBE3 aqueous solution in a place where it had been cooled to -40 ° C in advance, and freezing was performed while maintaining the shelf temperature at -40 ° C. . The frozen product was then freeze-dried to obtain a porous body.

Example 2
[Differential temperature measurement in each freezing step]
For each of “A” to “Ca”, the water surface liquid temperature at the center of the circle in the container as the liquid temperature in the solution farthest from the cooling side in the solution (non-cooled surface liquid temperature) The liquid temperature at the bottom of the container was measured as the liquid temperature closest to the side (cooling surface liquid temperature).
As a result, profiles of respective temperatures and their differential temperatures are as shown in FIGS. 1 to 6.
From this Fig. 1, Fig. 2, Fig. 3, the liquid temperature falls below 0 ° C, which is the melting point, in the shelf temperature -10 ° C setting section (before lowering to -20 ° C) for "A", "I" and "U". In this state, it is understood that freezing is not occurring (non-freezing / supercooling). Moreover, in this state, the temperature difference between the cooling surface liquid temperature and the non-cooling surface liquid temperature was 2.5 ° C. or less. Thereafter, by further lowering the shelf temperature to -20 ° C, the timing at which the liquid temperature rapidly rises to around 0 ° C is confirmed, and it can be seen that the heat of solidification is generated and freezing is started here. It was also confirmed that ice formation actually started at that timing. Thereafter, the temperature passes for a certain time around 0 ° C. Here, a mixture of water and ice was present. The temperature drop starts again from 0 ° C at this time, but at this time, the liquid portion disappears and it is ice. Therefore, the temperature being measured is the solid temperature inside ice, that is, not the liquid temperature.

  Below, about “A”, “I” and “U”, the differential temperature when the non-cooling surface liquid temperature reaches the melting point (0 ° C), just before lowering the shelf temperature from −10 ° C to −20 ° C Describe the differential temperature and the differential temperature just before the heat of solidification is generated. The term "differential temperature immediately before" as used in the present invention refers to the highest temperature among the temperature differences detectable between 1 second and 20 seconds before the event.

"A"
Differential temperature when uncooled surface liquid temperature reaches melting point (0 ° C): 1.1 ° C
Differential temperature just before lowering from -10 ° C to -20 ° C: 0.2 ° C
Differential temperature just before heat of solidification: 1.1 ° C

"I"
Differential temperature when uncooled surface liquid temperature reaches melting point (0 ° C): 1.0 ° C
Differential temperature just before lowering from -10 ° C to -20 ° C: 0.1 ° C
Differential temperature just before the heat of solidification: 0.9 ° C

"U"
Differential temperature when uncooled surface liquid temperature reaches melting point (0 ° C): 1.8 ° C
Differential temperature just before lowering from -10 ° C to -20 ° C: 1.1 ° C
Differential temperature just before the heat of solidification: 2.1 ° C

  These are hereinafter referred to as "a freezing step / porous body having a small temperature difference".

In addition, the liquid temperature is cooled to 0 ° C or lower, which is the melting point, by rapidly cooling the cabinet plate at -40 ° C or -60 ° C from Figs. In the state (non-freezing state / supercooling state), the differential temperature is 4 ° C. or more, and it can be seen that the differential temperature is large just before the heat of solidification is generated.
The differential temperatures when the non-cooling surface liquid temperature reaches the melting point (0 ° C.) and the differential temperatures immediately before the heat of solidification are described below for “D”, “E”, and “F”.

"D"
Differential temperature when uncooled surface liquid temperature reaches melting point (0 ° C): 5.4 ° C
Differential temperature just before the heat of solidification: 4.2 ° C

"O"
Differential temperature when the uncooled surface liquid temperature reaches the melting point (0 ° C): 6.9 ° C
Differential temperature just before solidification heat generation: 5.3 ° C

"Ca"
Differential temperature when the uncooled surface liquid temperature reaches the melting point (0 ° C): 5.9 ° C
Differential temperature just before the heat of solidification: 3.3 ° C

  These are hereinafter referred to as "a freezing step / porous body having a large differential temperature".

[Example 3]
[Low-temperature freezing step and drying step with 1% by mass ethanol-containing solution]
Pour CBE3 aqueous solution containing 1% (w / w) ethanol into a PTFE thick / cylindrical container and an aluminum glass plate / cylindrical container and use a cooling shelf in a vacuum freeze dryer (TF5-85ATNNN: Takara Seisakusho) The CBE3 aqueous solution was cooled from the bottom. The melting point is −0.4 ° C. by setting the final concentration to 1% by mass ethanol-containing aqueous solution. The melting point change in the ethanol / water concentration ratio was calculated from the document "Pickering SU: A Study of the Properties of some Strong Solutions. J. Chem. Soc. London 63 (1893) 998-1027".
The combination of the container, the final concentration of the CBE3 aqueous solution, the amount of liquid, and the setting of the shelf plate temperature at this time was prepared as described below.

"Aa" PTFE thick cylindrical container, final concentration 4% by mass of CBE3 aqueous solution, final concentration 1% ethanol by ethanol, amount of aqueous solution 4 mL. Set the shelf temperature to -10 ° C, cool at -10 ° C for 1 hour, then at -20 ° C for 2 hours, then at -40 ° C for 3 hours and finally at -50 ° C for 1 hour. The The frozen product is then vacuum dried at -20 ° C for 24 hours after returning the shelf temperature to -20 ° C, and after 24 hours, the shelf temperature is raised to 20 ° C while vacuum drying is continued. After vacuum drying was further carried out at 20 ° C. for 48 hours until the degree of vacuum was sufficiently lowered, it was removed from the vacuum lyophilizer. A porous body was thereby obtained.

"Good" Aluminum glass plate / cylindrical container, final concentration 4% by mass of aqueous solution of CBE3, final concentration of ethanol 1% by mass, amount of aqueous solution 4 mL. Set the shelf temperature to -10 ° C, cool at -10 ° C for 1 hour, then at -20 ° C for 2 hours, then at -40 ° C for 3 hours and finally at -50 ° C for 1 hour. The The frozen product is then vacuum dried at -20 ° C for 24 hours after returning the shelf temperature to -20 ° C, and after 24 hours, the shelf temperature is raised to 20 ° C while vacuum drying is continued. After vacuum drying was further carried out at 20 ° C. for 48 hours until the degree of vacuum was sufficiently lowered, it was removed from the vacuum lyophilizer. A porous body was thereby obtained.

[Differential temperature measurement in freezing step of 1% by mass ethanol-containing solution]
For “A” and “B”, the liquid surface temperature of the center of the circle in the container as the liquid temperature (uncooled surface liquid temperature) farthest from the cooling side in the solution, and on the cooling side in the solution The liquid temperature at the bottom of the container was measured as the closest liquid temperature (cooling surface liquid temperature). In addition, since 1 mass% ethanol becomes a solvent here, solvent melting | fusing point becomes -0.4 degreeC. The melting point change in the ethanol / water concentration ratio was calculated from the document "Pickering SU: A Study of the Properties of some Strong Solutions. J. Chem. Soc. London 63 (1893) 998-1027".

As a result, profiles of respective temperatures and their differential temperatures are as shown in FIG. 7 and FIG.
From FIGS. 7 and 8, in "A" and "B", the liquid temperature is below the melting point of -0.4.degree. C. in the shelf temperature -10.degree. C. setting section, and freezing does not occur in that state (not frozen)・ It turns out that it is a supercooling state. Moreover, in this state, the temperature difference between the cooling surface liquid temperature and the non-cooling surface liquid temperature was 2 ° C. or less. After that, by further lowering the shelf temperature to -20 ° C, the timing at which the liquid temperature rapidly rises to around -0.4 ° C was confirmed, where the heat of solidification was generated and freezing was started. I understand. It was also confirmed that ice formation actually started at that timing. Thereafter, the temperature passes for a certain time around -0.4 ° C. Here, a mixture of water and ice was present. The temperature drop starts again from 0 ° C at this time, but at this time, the liquid portion disappears and it is ice. Therefore, the temperature being measured is the solid temperature inside ice, that is, not the liquid temperature.

  Below, the difference temperature when the non-cooling surface liquid temperature reaches the melting point (-0.4 ° C) and the difference just before lowering the shelf temperature from -10 ° C to -20 ° C Describe the temperature and the temperature difference just before the heat of solidification.

"Aa"
Differential temperature when uncooled surface liquid temperature reaches melting point (-0.4 ° C): 0.8 ° C
Differential temperature just before lowering from -10 ° C to -20 ° C: 0.3 ° C
Differential temperature just before solidification heat generation: 0.8 ° C

"good"
Differential temperature when uncooled surface liquid temperature reaches melting point (-0.4 ° C): 1.3 ° C
Differential temperature just before lowering from -10 ° C to -20 ° C: 0.0 ° C
Differential temperature just before the heat of solidification: 1.3 ° C

  As a result of these, like "A" and "I", it was able to be manufactured as "a freezing process and a porous body with small temperature difference".

Example 4
[Evaluation of structural uniformity of porous material]
Evaluation of the porous structure uniformity of CBE3 porous bodies obtained by "A", "I", "U", "E", "O", "A", "A" and "I" did. The obtained porous body is thermally crosslinked at 160 ° C. for 20 hours to be insolubilized, and then swollen with physiological saline for a sufficient time. Thereafter, frozen tissue sections were prepared with a microtome, and HE (Hematoxylin Eosin)) stained specimens were prepared. The obtained sample images are shown in FIGS. When a two-dimensional Fourier transform (FFT) is applied to the 1.5 mm square field of view in the obtained sample image by the image analysis software ImageJ, a two-dimensional Fourier transform image shown in FIGS. When the two-dimensional Fourier transform confirms nonuniform orientation in the preparation, a strong power spectrum is observed particularly in the axial direction. This non-uniformity evaluation method is based on the non-patent document “Concrete Engineering Annual Proceedings, vol.22, No.1, 2000, p. 211, Basic research on evaluation method of soiling of concrete by two-dimensional Fourier transform, The method known by Kanematsu Studies, Kitagaki Kama, Noguchi Takafumi, Tomozawa Fuminori, etc. is applied to uniformity evaluation of porous bodies.

  Furthermore, in the present invention, the presence or absence of the power spectrum in the axial direction is defined by taking a line profile at the lower end of the y-axis of the two-dimensional Fourier transform image. Specifically, a graph was created in which the luminance values around the x-axis coordinate were averaged and plotted from the lower end of the y-axis to a width of 1/10 of the image pixel size. For example, the line profile was measured by the software “Gwyddion 2.31” as a 51 pixel wide line profile from a 512 pixel × 512 pixel two-dimensional Fourier transform image. Each line profile is shown in FIGS. Thus, assuming that the variance of the base of the line profile is σ, if a peak of 2.0σ or more is detected in the central portion (x of the line profile at half of the maximum value), the power spectrum in the axial direction is obtained. Was determined to exist. Inhomogeneous porous body in the presence of this power spectrum. In the case where there was no power spectrum, it was regarded as a uniform porous body.

  As a result, it was found that "A", "I", "U", "A" and "I", which are freezing processes and porous bodies with small temperature difference, are uniform porous bodies. In addition, as shown in Examples 2 and 3, the differential temperatures immediately before the heat of solidification in the production process of these porous bodies are respectively 1.1 ° C, 0.9 ° C, 2.1 ° C, 0.8 ° C, and 1. It is 3 ° C.

  On the other hand, it was found that “D” (2.0σ), “O” (6.0σ) and “Ca” (3.5σ), which are freezing processes and porous bodies with large temperature differences, are non-uniform porous bodies The As shown in Example 2, the differential temperatures immediately before the heat of solidification in the process of producing the porous body are 4.2 ° C., 5.3 ° C. and 3.3 ° C., respectively.

  Therefore, the temperature of the solution farthest from the cooling side in the solution (supercooled or unfreezed state below the melting point of the solvent (0 ° C for water, -0.4 ° C for 1% by mass aqueous ethanol)) Including a step in which the difference between the uncooled surface liquid temperature) and the liquid temperature (cooling surface liquid temperature) closest to the cooling side within the solution is within 2.5 ° C., and the temperature difference immediately before the heat of solidification is 2.5 It was found that a uniform porous body was obtained by setting the temperature to within ° C.

[Example 5] Freezing step with small temperature difference-Preparation of small temperature block from porous material (pulverization and crosslinking of porous material)
The CBE3 porous bodies of "A" and "A" obtained in Example 1 (differential temperature measurement is Example 2) were ground using a New Power Mill (Osaka Chemical, New Power Mill PM-2005). The grinding was performed by grinding for 1 minute x 5 times at s maximum rotation speed for a total of 5 minutes. The obtained pulverized material was sized with a stainless steel sieve to obtain 25 to 53 μm, 53 to 106 μm, and 106 μm to 180 μm uncrosslinked blocks. Thereafter, thermal crosslinking was carried out at 160 ° C. under reduced pressure (crosslinking time was carried out 6 kinds of 8 hours, 16 hours, 24 hours, 48 hours, 72 hours and 96 hours) to obtain a sample CBE 3 block. Hereinafter, the porous body-derived block of “A” crosslinked for 48 hours is E, and the porous body-derived block of “A” crosslinked for 48 hours is F. That is, E and F are small temperature difference blocks made from a freezing process and a porous body with small temperature difference. In addition, since the difference in crosslinking time does not affect the performance in the evaluation of the present invention, the one that has been crosslinked for 48 hours is used as a representative here.

Comparative Example 2 Preparation of a large block of differential temperature from a porous body and a freezing step having a large differential temperature (pulverization and crosslinking of the porous body)
The CBE3 porous bodies of "D", "O" and "K" obtained in Comparative Example 1 (differential temperature measurement in Example 2) were crushed by New Power Mill (Osaka Chemical, New Power Mill PM-2005) . The grinding was carried out at a maximum rotation speed for 1 minute x 5 times for a total of 5 minutes. The obtained pulverized material was sized with a stainless steel sieve to obtain 25 to 53 μm, 53 to 106 μm, and 106 μm to 180 μm uncrosslinked blocks. Then, thermal crosslinking was performed at 160 ° C. under reduced pressure (the crosslinking time was 24 hours and 48 hours) to obtain a sample CBE3 block. Thereafter, the porous body-derived block of "D" crosslinked for 48 hours is A, the porous body-derived block of "O" crosslinked for 48 hours B, porous for "K" crosslinked for 48 hours The body-derived block is called C. That is, A, B, and C are large-differential-temperature blocks made from a freezing process and a porous body having a large differential temperature. In addition, since the difference in crosslinking time does not affect the performance in the evaluation of the present invention, the one that has been crosslinked for 48 hours is used as a representative here.

[Example 6] Preparation of mosaic cell mass using small temperature difference block (hMSC)
Human bone marrow-derived mesenchymal stem cells (hMSC) growth medium (Takara Bio: MSCGM BulletKit ^TM) at was adjusted to 100,000 cells / mL, the CBE3 block 53-106μm prepared in Example 5 and 0.1 mg / mL After adding 200 μL, solubilize 200 μL on a Sumilon cellite X 96 U plate (Sumitomo Bakelite, U-shaped bottom), centrifuge (600 g, 5 minutes) in a table-top plate centrifuge, and allow to stand for 24 hours. A mosaic cell mass consisting of spherical, CBE3 blocks and hMSC cells was prepared (0.001 μg block per cell). In addition, since it produced in the U-shaped plate, this mosaic cell mass was spherical. Also, this could be created in the same manner as above for either E or F block.

Comparative Example 3 Preparation of Mosaic Cell Mass Using Differential Warm Large Block (hMSC)
Human bone marrow-derived mesenchymal stem cells (hMSC) growth medium (Takara Bio: MSCGM BulletKit ^TM) at was adjusted to 100,000 cells / mL, the CBE3 block 53-106μm prepared in Comparative Example 2 and 0.1 mg / mL After adding 200 μL, solubilize 200 μL on a Sumilon cellite X 96 U plate (Sumitomo Bakelite, U-shaped bottom), centrifuge (600 g, 5 minutes) in a table-top plate centrifuge, and allow to stand for 24 hours. A mosaic cell mass consisting of spherical, CBE3 blocks and hMSC cells was prepared (0.001 μg block per cell). In addition, since it produced in the U-shaped plate, this mosaic cell mass was spherical. Also, this could be created in the same manner as above for any of A, B and C blocks.

[Example 7] Preparation of mosaic cell mass using small temperature difference block (hMSC + hECFC)
Human vascular endothelial progenitor cells (hECFC) are adjusted to 100,000 cells / mL in growth medium (Lonza: EGM-2 + ECFC serum supplement), and the CBE3 block 53-106 μm prepared in Example 5 is 0.05 mg / mL. After adding 200 μL to Sumilongertite X96U plate, centrifuge (600 g, 5 minutes) in a table-top plate centrifuge, and stand for 24 hours, and flat, mosaic cell block consisting of ECFC and CBE3 block Was produced. Thereafter, the medium was removed, human bone marrow-derived mesenchymal stem cells (hMSC) growth medium (Takara Bio: MSCGM BulletKit ^TM) at was adjusted to 100,000 cells / mL, CBE3 block prepared in Example 5 53-106Myuemu Was added to a concentration of 0.1 mg / mL, and then 200 μL of hECFC mosaic cell mass was seeded on a Sumilongertite X96U plate, centrifuged (600 g, 5 minutes) using a table-top plate centrifuge, and allowed to stand for 24 hours. A 1 mm diameter spherical cell mass consisting of hMSC, hECFC and CBE3 blocks was prepared. Also, this could be created in the same manner as above for either E or F block.

Comparative Example 4 Preparation of Mosaic Cell Mass Using Differential Warm Large Block (hMSC + hECFC)
Human vascular endothelial precursor cells (hECFC) are adjusted to 100,000 cells / mL in growth medium (Lonza: EGM-2 + ECFC serum supplement), and the CBE3 block 53-106 μm prepared in Comparative Example 2 is 0.05 mg / mL. After adding 200 μL to Sumilongertite X96U plate, centrifuge (600 g, 5 minutes) in a table-top plate centrifuge, and stand for 24 hours, and flat, mosaic cell block consisting of ECFC and CBE3 block Was produced. Thereafter, the medium was removed, human bone marrow-derived mesenchymal stem cells (hMSC) growth medium (Takara Bio: MSCGM BulletKit ^TM) at was adjusted to 100,000 cells / mL, CBE3 block 53-106μm prepared in Comparative Example 2 Was added to a concentration of 0.1 mg / mL, and then 200 μL of hECFC mosaic cell mass was seeded on a Sumilongertite X96U plate, centrifuged (600 g, 5 minutes) using a table-top plate centrifuge, and allowed to stand for 24 hours. A 1 mm diameter spherical cell mass consisting of hMSC, hECFC and CBE3 blocks was prepared. Also, this could be created in the same manner as above for any of A, B and C blocks.

[Example 8] Fusion of mosaic cell mass (hMSC) using a small temperature differential block On the second day of the mosaic cell mass prepared in Example 6 (using the CBE 3 block of the present invention), 5 pieces of Sumilongertite X96U plate Arranged in, and cultured for 24 hours. As a result, it was revealed that the mosaic cell clusters are naturally fused by the cells arranged at the outer peripheral part being connected between the mosaic cell clusters. Also, this could be created in the same manner as above for either E or F block.

[Example 9] Fusion of mosaic cell mass (hMSC + hECFC) using a small temperature differential block [0172] Five day 2 mosaic cell mass (using the CBE 3 block of the present invention) prepared in Example 7 were treated with a Sumilongertite X96U plate Arranged in, and cultured for 24 hours. As a result, it was revealed that the mosaic cell clusters are naturally fused by the cells arranged at the outer peripheral part being connected between the mosaic cell clusters. Also, this could be created in the same manner as above for either E or F block.

[Comparative Example 5] Fusion of mosaic cell mass (hMSC) using a large temperature difference block Two day-old mosaic cell mass (derived from CBE 3 block for comparison) prepared in Comparative Example 3 in a Sumilongertite X96U plate Then, the cells were cultured for 24 hours. As a result, it was revealed that the mosaic cell clusters are naturally fused by the cells arranged at the outer peripheral part being connected between the mosaic cell clusters. Also, this could be created in the same manner as above for any of A, B and C blocks.

Comparative Example 6 Fusion of Mosaic Cell Mass (hMSC + hECFC) Using Differential Warm Large Block Five days from the second-day mosaic cell mass (derived from CBE3 block for comparison) prepared in Comparative Example 4 in a Sumilongertite X96U plate Then, the cells were cultured for 24 hours. As a result, it was revealed that the mosaic cell clusters are naturally fused by the cells arranged at the outer peripheral part being connected between the mosaic cell clusters. Also, this could be created in the same manner as above for any of A, B and C blocks.

[Example 10] Transplantation of mosaic cell mass using CBE3 block A mouse was a NOD / SCID (Charles River) male, 4 to 6 weeks old. Under anesthesia, the hair of the mouse abdomen is removed, a cut is made in the upper abdomen subcutaneously, scissors are inserted from the cut, and after the skin is peeled off from the muscle, Example 8, Example 9, Comparative Example 5, and Comparative Example The hMSC mosaic clumps prepared in 6 and hMSC + hECFC mosaic clumps were scooped with tweezers, and transplanted subcutaneously about 1.5 cm from the incision to the lower abdomen, and the incision of the skin was sutured.

Example 11 Collection of Mosaic Cell Mass Using CBE3 Block Dissection was performed 2 weeks after transplantation. The skin of the abdomen was removed, and the skin with the mosaic cell mass attached was cut into a square size of about 1 square cm. If the mosaic clumps were also attached to abdominal muscles, they were collected along with the muscles.

[Example 12] Specimen analysis Tissue sections were prepared for skin fragments to which mosaic cell masses were attached and mosaic cell masses before transplantation. The skin was immersed in 4% by mass paraformaldehyde and formalin fixation was performed. Thereafter, they were embedded in paraffin, and tissue sections of the skin containing mosaic cell masses were prepared. The sections were subjected to HE staining (hematoxylin and eosin staining).

  The HE specimens two weeks after the mosaic cell mass transplanted in Example 10 are shown in FIGS. The magnified image of 3 individuals is shown for mosaic cell clusters of each pattern, and the arrows point to blood vessels. The hMSC mosaic clumps of A and B are shown for 3 individuals, of which there are 6 individuals. The number of cells is the total number of living cells in the image, and the number of blood vessels is a value obtained by converting the number of blood vessels in each image into an area. The CV value is the ratio of the standard deviation of three or six mosaic cell clusters using each block divided by the average value, and indicates that the larger the numerical value, the greater the variation among individuals. Moreover, the summary of those results is shown in the following Tables 1-4.

The results with hMSC mosaic cell mass using each block are as follows.
FIG. 13 shows the results of hMSC mosaic cell mass using A and B large temperature differential blocks. In the large temperature differential block A, the average cell number was 122 cells and the average number of blood vessels was 12 / mm ² , and the CV values of the respective cells were 59% and 137%. The B cells had a mean cell number of 119 cells and a mean number of blood vessels of 10 cells / mm ² , and their CV values were 31% and 155%, respectively. From the section images, it can be seen that there is variation among mosaic cell masses.

FIG. 14 shows the results of hMSC mosaic cell mass using a C large differential heat block. In the C large differential heat block, the average number of cells was 122 cells, the average number of blood vessels was 17 / mm ² , and the CV value of each was 32% and 125%. Also from the section images, it can be seen that there is a variation between mosaic cell masses.

FIG. 15 shows the results with hMSC mosaic cell mass using E and F differential temperature small blocks. In the large temperature differential block E, the average cell number was 256 cells, the average number of blood vessels was 58 / mm ² , and the CV value of each was 7% and 10%. In the F block, the average number of cells was 194 cells, the average number of blood vessels was 54 / mm ² , and the CV values were 21% and 89%, respectively. Also from the section image, it can be seen that there is little variation among the mosaic cell masses.

The results with hMSC + hECFC mosaic cell mass using each block are as follows.
FIG. 16 shows the results with hMSC + hECFC mosaic cell mass using A and B large temperature differential blocks. In the A large differential heat block, the average number of cells was 84 cells, the average number of blood vessels was 34 / mm ² , and the CV values were 70% and 173%, respectively. In the B block, the average cell number is 158 cells, the average number of blood vessels is 51 / mm ² , and the CV values are 39% and 87%, respectively. It can be seen that there is variation.

FIG. 17 shows the results with hMSC + hECFC mosaic cell mass using E and F differential temperature small blocks. In the large temperature differential block E, the average cell number was 202 cells, the average number of blood vessels was 122 / mm ² , and the CV value of each was 29% and 17%. In the F block, the average number of cells was 171 cells, the average number of blood vessels was 109 / mm ² , and the CV values were 29% and 29%, respectively. Also from the section images, it can be seen that there is no variation among the mosaic cell masses.

Although it is as having put together in Table 1-Table 4 as a whole, in the case of using a large temperature control block, it turns out that the individual difference between mosaic cell masses is large and it is scattered. It can be seen that the average cell number and the average number of blood vessels are not significantly different depending on the type of the large temperature differential block.
On the other hand, in the case of using the small temperature differential block, it is understood that the individual difference between mosaic cell masses is small and the variation is small. The average cell number and the average number of blood vessels also show significantly higher values than the large temperature differential block, and it can be said that the small temperature differential block is more likely to form blood vessels and allows many cells to survive. As shown in Example 4, since the porous body obtained in the freezing step with small temperature difference was uniform in structure as shown in Example 4, in the small temperature difference block made from the porous body, It is considered that the variation in in vivo performance is reduced. Furthermore, it was also unexpectedly found that it also contributes to the ability to form blood vessels and the viability of cells. In addition, in order to exert the performance of the transplanted cells, it is very important to enhance both the ability to form blood vessels and the viability of the transplanted cells. The quality body was showing high effect.

[Example 13] Calculation of proportion and concentration of ECFC in hMSC + hECFC mosaic cell mass The mosaic cell mass using representative E among Example 7 is sectioned for hECFC staining with CD31 antibody (EPT Anti CD31 / PECAM 1) Immunostaining with a kit using DAB coloring (Dako LSAB2 kit universal K0673 DAKO LSAB2 kit / HRP (DAB) rabbit / mouse primary antibody both). Using the image processing software ImageJ and a staining method with a CD31 antibody, the ratio of the area of hECFC (cells of the vascular system) in the central part was determined. In addition, the "central part" said here is what was defined above.

As a result, the percentage of the area of hECFC (the cells of the vascular system) in the central part of the mosaic cell mass of representative E of Example 7 was 98%. Furthermore, the cell density of hECFC present in the central part was calculated by superimposing the staining with the above-described CD31 antibody and HE staining (hematoxylin and eosin staining) for the representative E mosaic cell mass of Example 7. The cell density of the central vasculature can be determined by actually counting the number of cells of the sliced specimen and dividing the number of cells by volume. First, using Photoshop, the above two images were superimposed, and the number of HE stained cell nuclei overlapped with the staining with the CD31 antibody was counted to calculate the number of cells. On the other hand, the volume was determined by calculating the area of the central part using ImageJ and multiplying 2 μm of the thickness of the sliced sample. As a result, the cell number of hECFC (cells of the vascular system) in the central part of the representative E mosaic cell mass of Example 7 was 2.3 × 10 ⁻⁴ cells / μm ³ .

Claims (23)

The y-axis lower end of the two-dimensional Fourier transform image of the two-dimensional Fourier transform image obtained by applying the two-dimensional Fourier transform to the 1.5 mm square field of view of the cross-sectional structure of the biocompatible polymer porous body From the above, when the line profile is created by averaging the luminance values around the x-axis coordinates and creating a line profile plotted on the y-axis with a width of 1/10 of the image pixel size, x on the line profile is half of the maximum value It is a bioaffinitive polymeric porous body which does not have a peak in (a), Comprising: The bioaffinitive polymeric porous body which has a void | hole of 10 micrometers or more and 500 micrometers or less . Assuming that the variance of the base of the line profile is σ, there is no peak when a peak of 2.0 σ or more is not detected at half of the maximum value of x on the line profile of the two-dimensional Fourier transform image. The biocompatible polymer porous body according to claim 1. Biocompatible polymers are gelatin, collagen, elastin, fibronectin, pronectin, laminin, tenascin, fibrin, fibroin, entactin, thrombospondin, retronectin, polylactic acid, polyglycolic acid, lactic acid / glycolic acid copolymer, hyaluronic acid, glycoic acid The biocompatible polymer porous body according to claim 1 or 2, which is a Saminoglycan, proteoglycan, chondroitin, cellulose, agarose, carboxymethylcellulose, chitin or chitosan. The biocompatible polymer porous body according to any one of claims 1 to 3, wherein the biocompatible polymer is a recombinant gelatin. Recombinant gelatin is
Formula: A-[(Gly-X-Y) n] m-B
(Wherein, A represents any amino acid or amino acid sequence, B represents any amino acid or amino acid sequence, n Xs each independently represent any one of amino acids, n Ys each independently represent an amino acid) And n represents an integer of 3 to 100, and m represents an integer of 2 to 10. The n Gly-XYs may be the same or different. The biocompatible polymer porous body according to Item 4. Recombinant gelatin is
(A) a protein having the amino acid sequence set forth in SEQ ID NO: 1;
(B) a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1 and having biocompatibility; or (c) described in SEQ ID NO: 1 A protein having an amino acid sequence having 80% or more homology with the amino acid sequence of
The biocompatible polymer porous body according to claim 4, which is any one of the above. The bioaffinity macroporous material according to claim 4, wherein the recombinant gelatin is a protein having an amino acid sequence having 90% or more homology with the amino acid sequence of SEQ ID NO: 1 and having biocompatibility. body. The bioaffinity macroporous material according to claim 4, wherein the recombinant gelatin is a protein having an amino acid sequence having 95% or more homology with the amino acid sequence of SEQ ID NO: 1 and having biocompatibility. body. The biocompatible polymer porous body according to any one of claims 1 to 8, wherein the biocompatible polymer in the porous body is crosslinked by heat, ultraviolet light or an enzyme. A biocompatible polymer block obtained by grinding the biocompatible polymer porous body according to any one of claims 1 to 9. A cell structure comprising the biocompatibility polymer block according to claim 10 and at least one type of cell, wherein a plurality of biocompatibility polymer blocks are arranged in a gap between a plurality of cells. The cell structure according to claim 11, wherein a size of one biocompatible polymer block is 20 μm or more and 200 μm or less. The cell structure according to claim 11 or 12, wherein the size of one biocompatible polymer block is 50 μm or more and 120 μm or less. The cell structure according to any one of claims 11 to 13, which has a thickness or a diameter of 400 μm to 3 cm. The cell structure according to any one of claims 11 to 14, which contains 0.0000001 μg or more and 1 μg or less of a biocompatible polymer block per cell. The cell structure according to any one of claims 11 to 15, comprising an angiogenic factor. The cell structure according to any one of claims 11 to 16, wherein the cell is a cell selected from the group consisting of a pluripotent cell, a somatic stem cell, a precursor cell and a mature cell. The cell structure according to any one of claims 11 to 17, wherein the cells are only non-vascular cells. 18. A cell structure according to any one of claims 11 to 17, wherein the cells comprise both non-vascular and vascular cells. 20. The cell structure according to claim 19, wherein in the cell structure, the area of cells in the central vasculature has a larger area than the area of cells in the peripheral vasculature. 21. The cell structure according to claim 20, having a region in which the percentage of cells in the central vasculature is 60% to 100% with respect to the total area of the cells in the vasculature. The cell structure according to any one of claims 19 to 21, which has a region in which the cell density of the central vasculature is 1.0 × 10 ^-4 cells / μm ³ or more. Cell structure. The cell structure according to any one of claims 19 to 22, which is vascularized inside the cell structure.